Project Summary/Abstract Lung cancer has been the leading cause of cancer-related deaths in the United States for several decades, yet it has experienced only incremental improvements in survival and consistently receives disproportionately low funding for research. Although the emergence of targeted therapies has significantly improved survival in a fraction of patients with non-small cell lung cancer (NSCLC), these patients invariably acquire resistance. The majority of other patients, those without actionable mutations, do not have treatment options besides standard chemotherapy regimens. Recently, therapeutic antibodies against immune checkpoint protein programmed death 1 (PD-1) receptor have been approved for NSCLC. Though they provide appreciable clinical benefit, approximately 80% of patients fail to respond, and the prognostic value of PD-L1 expression remains unclear. Thus, there is a clear need for a better understanding of the tumor immune microenvironment in NSCLC. EphA2 receptor tyrosine kinase is overexpressed in therapy-resistant NSCLC harboring EGFR or KRAS mutations. Inhibition of EphA2 induces apoptosis in NSCLC cells in vitro and decreases tumor burden in analogous mouse models. Preliminary data presented in this proposal suggests that EphA2 may upregulate PD-L2, but not PD-L1 expression, in NSCLC, contributing to immune evasion. This proposal aims to (1) determine the role of EphA2 on modulating PD ligand expression in NSCLC cells and subsequent anti-tumor T cell response and (2) evaluate the effect of genetic and pharmacologic inhibition of EphA2 on the tumor immune microenvironment in vivo, particularly T cell-mediated anti-tumor immunity. The effect of EphA2 in altering PD-L1/2 expression in therapy-resistant EGFR and KRAS mutant human NSCLC cell lines will be assessed by generating EphA2 loss of function using CRISPR/Cas9 technology and measuring changes in PD-L1/2 expression. EphA2?s role in mediating antigen-specific T cell response will be evaluated with in vitro functional co-culture assays, as well as in a syngeneic in vivo murine NSCLC model. Effects of PD-L1 and PD- L2 blockade will also be tested in these experiments, which include T cell-mediated cancer cell killing, T cell proliferation and apoptosis, cytokine production, and intratumoral T cell infiltration and activation. Additionally, the impact of genetic and pharmacologic inhibition of EphA2 on anti-tumor immunity in NSCLC will be investigated in transgenic and transplantation mouse models, including a novel model utilizing CRISPR/Cas9 technology. EGFR and KRAS mutant lung tumors will be induced in these mice, and the effects of EphA2 LOF or EphA2 kinase inhibitors on T cell infiltration and activation, tumor burden over time, and survival will be assessed. The success of this project will have far-reaching significance by advancing our understanding of tumor immunobiology, especially the role of PD-L2, and increasing the translational potential of new therapies for NSCLC.